LIGAND SPECIFICITY OF HUMAN THROMBOMODULIN - EQUILIBRIUM BINDING OF HUMAN THROMBIN, MEIZOTHROMBIN, AND FACTOR XA TO RECOMBINANT THROMBOMODULIN

Thrombomodulin is an endothelial glycoprotein that serves as a cofacto r for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human th rombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition bin ding assays with CV-1(18A) cells expressing cell surface recombinant h uman thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited I-125-diisopropyl fluorophosphate-thrombin binding with sim ilar affinity (K(d) = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). H owever, no binding inhibition was detected for meizothrombin S205A or human factor Xa (K(d) > 500 nM). In direct binding assays, I-125-label ed plasma thrombin and thrombin S205A bound to thrombomodulin with K(d ) values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. I-125-Labele d meizothrombin S205A and human factor Xa did not bind to thrombomodul in (K(d) > 500 nM). We also compared the ability of thrombin and facto r Xa to activate human recombinant protein C. The activation of recomb inant protein C by thrombin was greatly enhanced in the presence of th rombomodulin, whereas no significant activation by factor Xa was detec ted with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells we re used as the source of thrombomodulin. These results suggest that hu man meizothrombin and factor Xa are unlikely to be important thrombomo dulin-dependent protein C activators and that thrombin is the physiolo gical ligand for human endothelial cell thrombomodulin.